This proposal examines the molecular basis of differential gene expression in the simple eukaryotic organism, the yeast Saccharomyces cerevisiae. Specifically, the goal of this proposal is to understand two facets of the regulation of genes involved in yeast conjugation. Conjugation (mating) occurs between two distinct cell types of yeast, a and Alpha, and the process of mating is under both genetic and physiological controls. Expression of genes whose products are required for mating is regulated by alleles of a single genetic locus, the mating type locus (MATAlpha and MATa). In addition, peptide pheromones secreted by each cell type also affect expression of some genes required for mating. We have focused on a particular gene, STE3, which is required for mating only by Alpha cells. STE3 is regulated in two ways: RNA production from this gene requires the MATAlpha1 product of the Alpha mating type locus, and RNA production from STE3 increases in response to treatment of Alpha cells with the a cell pheromone, a factor. Our specific aims are to understand these two modes of control using a combination of genetic, molecular cloning, and biochemical approaches. The region of the STE3 gene that imparts the unique features of its regulation will be defined by in vitro mutagenesis. The role of the MATAlpha1 product will be investigated by determining both its subcellular location and whether it interacts directly with STE3 DNA sequences. Other potential regulators besides MATAlpha1 will be indentified by in vivo mutagenesis. Finally, the role of the STE3 product will be investigated. Preliminary evidence suggests that it is a component of the a-factor receptor. This view will be tested both by determining the subcellular location of STE3 product and whether a-factor binds to Alpha cells in a STE3-dependent manner.